This project is concerned with the role(s) of cAMP and related adrenergic drug mechanisms in aqueous humor dynamics. The proposed studies are focused on cAMP in ciliary processes (CP) and in trabecular tissues (TM). The research approach combines biochemical and molecular biology studies with measurements on aqueous humor dynamics in the living rabbit and monkey eye. Ciliary epithelial transports are postulated to be controlled by anion channels which are in turn controlled by cAMP/adenylyl cyclases (AC) with different regulation on basolateral and apical membranes. A new objective is to fully characterize the AC enzyme systems in TM tissue. Biochemical findings will be followed-up by in vivo experiments on outflow facility in monkeys, and the perfused human anterior segment in vitro. Specific objectives are: Cloning of different adenylyl cyclases in TM and CP tissue; regulation of AC by mechanisms beyond the receptor level especially by intracellular monovalent ions; to define a regulatory site on AC which binds compounds with the general structure XO2-OH; to develop more potent and selective inhibitors to distinguish different types of AC; to study in the primate eye, interactions of FSK with alpha-adrenergic agonists in relation to inflow and outflow mechanisms; a detailed and comprehensive biochemical study of trabecular cAMP/adenylyl cyclase systems in bovine and human trabecular tissue; and to determine facility responses in the perfused anterior segment system. These studies are of fundamental importance to understand the biochemistry and molecular pharmacology of aqueous humor formation by ciliary processes, and of facility changes in trabecular meshwork. They are relevant to the pathophysiology and the therapy of glaucoma. This project provided the first biochemical characterization of ACs with different regulation (such as a bicarbonate sensitive adenylate cyclase in fluid transporting tissues), and led to the discovery of a new regulatory site on AC, and the first compounds with selectivity for various types of AC enzymes. Therefore, this project also has wide implications for cAMP/adenylyl cyclase research in general.
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